The biosensor literature consists of numerous publications (patents, articles, and books) per year. A short description of the mode of operation of biosensors is given below. Then, several publications relevant to this patent are described, along with a comparison of their features to those of the present invention.
Biosensors consist of a biological interface (e.g., an enzyme or cell layer) coupled to a transducer (e.g., an oxygen electrode). The biological element gives selectivity and specificity for detecting specific analyte molecules. The interaction of the biological element with the analyte is measured by the physical transducer. In the case of electrochemical biosensors, the transducer is typically an electrode.
Electrochemical biosensors often have a membrane separating the electrode from the surrounding liquid. The relative rates of analyte transport to the electrode and electron transfer at the electrode affect the performance properties of the biosensor. The overall transport resistance is the sum of the resistances in the membrane and the stagnant liquid film adjacent to the membrane. If the liquid-film resistance is significant, the probe's response is dependent on the local liquid velocity, which is typically controlled by the rate at which the specimen is stirred. To prevent stirring dependency, the resistance of the membrane can be increased. However, this approach leads to slow probe response times. Thus, there is typically a tradeoff between the speed of the probe's response and the rate at which the liquid sample is stirred. For specimens that cannot be stirred (e.g., in situ measurements within highly viscous or semi-solid samples), conventional biosensors give slow or inaccurate readings.
Due to their extremely small size and rapid electron transfer, microbiosensors can provide both rapid response characteristics and independence of stirring rate. Thus, they are especially well-suited for applications that require rapid, repetitive measurements, especially where stirring may not be possible. Their extremely small tip size also allows accurate measurements within small specimens, measurements of concentration gradients with high resolution, and measurements inside of samples with minimal surface disturbance. Several ideal application areas are listed below:
Monitoring food quality and safety, such as the degree of freshness and the concentration of toxicants.
Measuring gradients within biofilms, and other small-scale ecosystems.
Monitoring the activity of single cells, such as neurotransmission events.
An electrochemical biosensor is developed in the world patent application WO/PCT 92/04438 by Eisenhardt and Christiensen from Radiometer A/S, Copenhagen, Denmark (1992). It consists of a working electrode and a reference electrode. The base part contains a working electrode in the form of platinum wire that has 250 .mu.m diameter. The laminated outer membrane consists of a 15 .mu.m thick protective layer. This microsensor is several centimeters in diameter and is similar in many aspects to the type E909, sold by Radiometer. Its main drawbacks, if compared with the present invention disclosure, are slower response times, sensitivity to stirring, and its use limited to liquid samples drawn from the process.
The antibody-antigen biosensor for determining lactate dehydrogenase-5 was devised by Risphon et al. (1993, U.S. Pat. No. 5,147,781). Antibodies were bound directly to an electrically conductive electrode. However, these antibodies are difficult and time consuming to prepare and their extremely high affinity makes the dissociation kinetics slow, which makes the sensor response slow.
A single-chip, planar shape receptor-based biosensor is described (1993) by Taylor et al., Arthur D. Little Inc., Cambridge, Mass., in U.S. Pat. No. 5,192,507. In particular, acetylcholine receptor and opiate receptor have been immobilized in a polymeric film made with bovine serum albumin, gelatin and glutaraldehyde. An in situ repetitive use has not been considered for these microelectronic biosensors. Another disadvantage of this type of biosensor is that an adequate method for fixation and sealing of the diffusion-limiting membrane around the electrode perimeter has not been developed (Alvarez-Icaza and Bilitewski, 1993). Moreover, this probe uses glutaraldehyde, which is known to be cytotoxic (Simmons and Kearney, 1993) and therefore unsafe for some biosensor applications.
"Ultrasmall" glucose sensors have been constructed for voltammetry and amperometry (Abe, Lau and Ewing, 1992; Kim, Scarnulis and Ewing, 1986) by using platinum deposited carbon ring microelectrodes with glucose oxidase. The 2-10 .mu.m sensing tip allowed average response times of 0.8 seconds. The detection limit is reported as 50.mu.M, and the linear range is up to 5 mM. Nevertheless, the amperometric measurements are carried out in a two-electrode mode, which is difficult to perform in situ. Another drawback is that the sensor produces a noisy signal, so that a copper mesh Faraday cage is required as an electromagnetic shield. Moreover, this microsensor design includes a mercury film, which is toxic. Other problems are the electrode fouling and the short stability span, of only "a few hours". The microbiosensor of this invention has proved a lower and better detection limit of less than 10.mu.M and good stability after multiple tests over a period of time, e.g., 6 consecutive months. The present application has low electrical noise. Both the reference and the working electrodes are situated inside the sensor case, behind an electrically insulating silicone film, and bathed in an electrolyte solution such as 1M KCl. The electrolyte also serves as an electrical shielding from the cathode. The signal from the microelectrode is therefore expected to have an extremely low noise and to be very stable, with a current drift less than 2% per day.
Other miniature enzymatic biosensors are made using carbon fibers, e.g. U.S. Pat. No. 5,186,808, by Yamaguchi and co-workers (1993) from Terumo K.K. Company, Tokyo, Japan. This particular graphite electrode has an electrical conductive substrate with a sectional area of less than 10.sup.-5 cm.sup.2, which means the electrode hole diameter is about 35.7 .mu.m. The major drawback of this patent is that the enzyme sensor is used in a three electrode cell. The use of three electrodes, some of which may not have microscopic tip dimensions, would make in situ measurements difficult or even impossible. By contrast, the present microbiosensor has all electrodes built in one case. In addition to greater convenience, this integration also results in reduced electrical noise levels. Also, the sensitivity reported by Yamaguchi et al. for glucose is low, 1 mM, when compared with the present microbiosensor.
Other sensors based on carbon fibers (Karube et al., 1993) or of solid-state type (Kawaguri et al., Matsushita Co., Japan, U.S. Pat. No. 5,171,689) report the use of 1, 4-benzoquinone or ferricyanide as electron mediators/oxidizing agents. Unfortunately, both 1, 4-benzoquinone and ferricyanide may be toxic. Benzoquinone toxicity has been proven, including for short term bacterial bioassays (Trevors and Basaraba, 1980), isolated rat hepatocytes (Nakagawa and Moldeus, 1992) or mice bone marrow cells (Neun et al., 1992; Larsson et al, 1986). Ferricyanide was shown to have an embryotoxic action (Besedina and Grin, 1987), is relatively toxic to mammalian cells (Lai et al, 1987) and may cause structural damage on the skeletal muscle (Duncan, 1989). These two toxic mediators could potentially leach into the sample.
An optical biosensor is reported (1992) by Morris and colleagues from Baxter Diagnostics Inc., Illinois, in the world patent application WO92/12413, to detect microorganisms in a blood culture bottle. This application, and many other biosensors using a fiber optic transducer, have the disadvantages of being subject to interference from ambient light (Luong et al., 1991), usually requiring high energy sources and often suffering from a narrow concentration range.
Microbial biosensors (e.g. Scheller, 1993; Lee et al., 1992; Karube and Suzuki, 1990) are yet another method, which incorporate a microorganism as sensing element and can measure the respiration activity (detected by an oxygen sensor) or electroactive metabolites, such as H.sub.2, CO.sub.2, NH.sub.3 and organic acids, secreted by the microorganisms. Although these sensors may exhibit a long shelf life and are more pH and temperature tolerant if compared to the enzyme probes, these microbial sensors have a longer response time, need more time to return to the base line and additional care must be taken to ensure selectivity (Karube and Nakanishi, 1994).
It is an object of the present invention to describe a microbiosensor having a needle-type, e.g., cylindro-conical configuration with a sensing tip aperture not greater than 25 (preferably 4) micrometers (.mu.m) and having the ability to be compatible with the specimen or host such that the outer protecting membrane that is utilized is non-virulent thereto.
Also of interest are the following U.S. Patents:
U.S. Pat. No. 4,680,268 describes an implantable biosensor and a method for sensing products. A closed chamber for containing oxygen to supply oxygen through a membrane for the enzymatic reaction is described. However, the sensor does not appear to be of the micro-type. The precise geometry and functional characteristics (e.g. life span, range and detection limit) are not specified.
U.S. Pat. No. 4,871,440 describes a biosensor which has a foundation electrode comprising of a working electrode, a reference electrode and a counter electrode arranged on a planar surface.
U.S. Pat. No. 5,120,420 describes a planar type biosensor where a biological sample solution is brought into contact with the inlet 10 of the biosensor while the air within the space 8 is rapidly discharged through the outlet 11.
U.S. Pat. No. 5,177,012 describes a biosensor containing immobilized Zymomonas mobilis cells for measuring glucose, fructose and sucrose.
U.S. Pat. No. 5,185,256 pertains to a biosensor where the electrode system is formed mainly of carbon and is integrally combined with a perforated body so that washing the electrode system is unnecessary. The planar electrode system is formed on a substrate and is primarily made of carbon in a perforated body having an enzyme and electron acceptor.
U.S. Pat. No. 5,223,124 is a monolayer needle electrode having a core platinum anode (2) situated inside a stainless steel reference cathode (4). The stainless steel tube has an outer diameter of 0.46 mm. The enzyme is immobilized onto angular surface A. The immobilization of a polypeptide such as an enzyme was performed by blending the enzyme in a polymeric matrix such as an aqueous polyurethane dispersion which is applied to the angular member A. The detection limit for glucose appears to be 2.4 mM. The biosensor is usable 5-24 hours.
U.S. Pat. No. 5,286,364 described an electrode for a biosensor, wherein the analyte sensing agent is an enzyme which is embedded in a polymer but with a number of its analyze recognition sites unblocked. FIG. 15 is a graph of the glucose concentration v. steady state current. The working electrode of the biosensor is used in a flow cell injection system, of the three-electrode type. The microbiosensor features a detection limit of approximately 50.mu.M with a response time of 1-2 minutes.
U.S. Pat. No. 5,288,636 describes a probe for glucose using a redox mediator of ferricyanide.
Other patents of interest are: U.S. Pat. Nos. 5,288,613; 5,356,786; 5,225,064; and 5,334,296.
In summary, the biosensor systems described previously are subject to one or more of the following drawbacks relative to the present invention: slower response times, stirring dependency, necessity of diluting the sample before measurement, narrower concentration range, use of potentially toxic electron mediators, high levels of electrical noise, and poor long-term stability.